John C. Khoo

Enhanced macrophage uptake of low density lipoprotein after self-aggregation.

Incubation of mouse peritoneal macrophages with native human low density lipoprotein (LDL) did not cause any significant storage of Intracellular cholesteryl esters. However, when the LDL was subjected to brief (30-second) vortexlng, It formed self-aggregates that were rapidly Ingested and degraded by macrophages, converting them to cholesteryl ester-rich foam cells. Such aggregates were as potent as acetyt-LDL In stimulating cholesterol esterlflcatlon in the macrophages. The degradation of LDL aggregates was strongly inhibited by cytochalasln B (85%), whereas degradation of native LDL was only weakly Inhibited (23%), suggesting that uptake occurred by phagocytosis rather than plnocytosls. Several lines of evidence suggest that the phagocytlc uptake depends, in part, upon the LDL receptor and not the acetyl-LDL receptor: 1) soluble, native LDL and 0-VLDL (but not acetyl-LDL) competed for uptake and degradation of LDL aggregates; 2) reductive methylatlon of LDL before vortexlng reduced the effect of the aggregates on degradation and cholesterol esterlflcatlon; 3) heparln, which Inhibits binding of native LDL to Its receptor, reduced the degradation of LDL aggregates. These studies show that self-aggregation of LDL markedly enhances Its uptake by macrophages, probably by phagocytosis and at least, In part, via the LDL receptor. Aggregates of LDL in the artery wall–either self-aggregates or mixed aggregates Including matrix components–may Induce foam cell formation and favor the formation of the fatty streak.

Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts.

Pure macrophage-derived foam cells (MFC) were isolated from the aortas of rabbits made atherosclerotic by balloon deendothelialization followed by diet-induced hypercholesterolemia. The MFC were isolated under sterile conditions using an enzymatic digestion procedure and discontinuous density gradient centrifugation. The purity of the MFC preparations was verified immunocytochemically with the macrophage specific monoclonal antibody RAM-11. MFC plated in medium containing 0.5% FCS for 24 h contained approximately 600 micrograms cholesterol per mg cell protein, 80% of which was esterified cholesterol. The MFC specifically degraded low density lipoprotein (LDL), acetyl-LDL, copper oxidized LDL, and beta-very low density lipoprotein (beta-VLDL) at rates comparable to mouse peritoneal macrophages (MPM) in 5-h assays. MFC within sections of the atherosclerotic lesions from the ballooned rabbits as well as the MFC isolated from the same lesions in the presence of antioxidants, exhibited positive immunoreactivity with polyclonal guinea pig antisera and mouse monoclonal antibodies directed against malondialdehyde-LDL, and 4-hydroxynonal-LDL. The MFC also exhibited the capacity to induce the oxidation of LDL at rates comparable to those exhibited by MPM and rabbit aortic endothelial cells. These data provide direct evidence that arterial wall macrophages express modified LDL receptors in vivo, contain epitopes found in oxidized-LDL and are capable of oxidizing LDL even when maximally loaded with cholesterol.

Isoenzymes of adenylate kinase in human tissue

Abstract A study of the isoenzymes of adenylate kinases (ATP:AMP phosphotransferases EC 2.7.4.3) from several tissues of human and rabbit indicated that there is a minimum of two sets of isoenzymes within an individual. Isoenzyme sets were distinguished by differences in inhibitions by AgNO3 and an antiserum against rabbit muscle adenylate kinase. The adenylate kinases from muscle, erythrocytes and brain were similar and form one set of isoenzymes. The adenylate kinases of liver, kidney, spleen and heart were similar and form another set of isoenzymes.

Stimulation of rat liver phosphorylase kinase by micromolar concentrations of Ca2

Phosphorylase kinase from rabbit skeletal muscle [l-3], cardiac muscle [3], insect flight muscle [4], guinea pig brain [5] and human platelets [6] has been shown to require micromolar concentrations of Ca*’ for optimal activity. Recently, we have shown that phosphorylase kinase from adipose tissue also has a Ca*+ requirement [7,8]. The results presented below show that phosphorylase kinase from rat liver requires Ca*+ for optimal activity. It was inhibited 75% by 0.2 mM EGTA [ethyleneglycol-his @-aminoethyl ether)-iii, N’-tetraacetic acid]. This inhibition was promptly and fully reversed by Ca*+. The possible involvement of liver phosphorylase phosphatase was ruled out by inclusion of fluoride (F -) and p-glycerolphosphate @-GP) in the assay system, which effectively blocked phosphatase activity.

An in vitro model to study adipose differentiation in serum-free medium☆

Abstract Adipose differentiation was studied in a teratoma-derived fibroadipogenic cell line (1246) cultured in serum-free medium. The addition of dexamethasone and 1-methyl-3-isobutylxanthine to the serum-free medium induced confluent 1246 cells to differentiate into adipocyte-like cells as evidenced by triglyceride accumulation and increased levels of lipolytic enzyme activities. Hormone-sensitive lipase activity measured 5 days after the addition of dexamethasone and 1-methyl-3-isobutylxanthine increased 17-fold and was activated by cAMP-dependent protein kinase. Neutral diglyceride lipase, monoglyceride lipase, and cholesterol ester hydrolase specific activities increased 23-, 75-, and 73-fold, respectively. Among these three activities, only cholesterol ester hydrolase was activated by cAMP-dependent protein kinase. Differentiated 1246 cells expressed receptors to lipolytic hormones as shown by the stimulation of glycerol release by epinephrine (8.6-fold), glucagon (2.2-fold), and adrenocorticotrophic hormone (5.5-fold). Heparin treatment of 1246 cells in serum-free medium resulted in the release of lipoprotein lipase activity into the culture medium. Thus, 1246 cells can serve as a model for the study of adipose differentiation under defined culture conditions since they are capable of growth and survival in the absence of serum while retaining their ability to differentiate into adipocytes.

Activation of hormone-sensitive lipase and phosphorylase kinase by purified cyclic GMP-dependent protein kinase

Cyclic GMP-dependent protein kinase, purified to homogeneity from bovine lung, was shown to activate hormone-sensitive lipase partially purified from chicken adipose tissue. The degree of activation was the same as that effected by cyclic AMP-dependent protein kinase although higher concentrations of the cyclic GMP-dependent enzyme were required (relative activities expressed in terms of histone H2b phosphorylation units). Activation by cyclic AMP-dependent protein kinase was completely blocked by the heat-stable protein kinase inhibitor protein from skeletal muscle but activation by the cyclic GMP enzyme was not inhibited. Lipase fully activated by cyclic AMP-dependent protein kinase showed no further change in activity when treated with cyclic GMP-dependent protein kinase. Lipase activated by cyclic GMP-dependent protein kinase was reversibly deactivated by purified phosphorylase phosphatase (from bovine heart); full activity was restored by reincubation with cyclic GMP and cyclic GMP-dependent protein kinase. Cholesterol esterase activity in the chicken adipose tissue fraction, previously shown to be activated along with the triglyceride lipase by cyclic AMP-dependent protein kinase, was also activated by cyclic GMP-dependent protein kinase. Crude preparations of hormone-sensitive triglyceride lipase from human or rat adipose tissue and cholesterol esterase from rat adrenal were also activated by cyclic GMP-dependent protein kinase. Purified phosphorylase kinase (rabbit skeletal muscle) was also shown to be activated by cyclic GMP-dependent protein kinase. The present results, together with those of other workers on histone phosphorylation, suggest that the substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinase may be similar. This is discussed in the light of a model recently proposed with regard to the relationship between the subunit structures of the two kinases. The physiologic significance of the findings remains to be established.

Polymerase Chain Reaction–Based Method for Quantifying Recruitment of Monocytes to Mouse Atherosclerotic Lesions In Vivo Enhancement by Tumor Necrosis Factor-α and Interleukin-1β

Abstract —The critical role of monocyte recruitment in atherogenesis has been appreciated for some time. However, until recently, there have been no sufficiently sensitive methods for measuring rates of monocyte recruitment to the arterial wall in vivo. We have developed a novel highly sensitive method, based on the polymerase chain reaction, for quantitatively tracking DNA-marked monocytes and have adapted it for use in mice. We use the uniquely male gene, Sry , on the Y chromosome as a gene marker. We transfuse monocytes from a male donor into a congenic female mouse, euthanize the mouse after 24 to 48 hours, and then quantify the arterial uptake of monocytes by quantitative polymerase chain reaction. This study describes the techniques used and their sensitivity and reproducibility and demonstrates the approach by assessing the effects of cytokines. In control low density lipoprotein receptor–negative mice, monocyte recruitment decreased slightly but significantly as lesions progressed. Intraperitoneal injection of a combination of tumor necrosis factor-α and interleukin-1β more than doubled the rate of monocyte recruitment into developing lesions. However, the response to the cytokines was much greater in younger mice with less advanced lesions than in older animals with more advanced lesions.

Stimulation of a neutral cholesteryl ester hydrolase by cAMP system in P388D1 macrophages.

Cholesteryl ester laden foam cells in atherosclerotic lesions derive, in part, from macrophages. Mobilization of stored cholesteryl esters involves hydrolysis by a neutral cholesteryl ester hydrolase. Incubation of intact P388D1 macrophages with dibutyryl cAMP in the presence of 1-methyl-3-isobutylxanthine resulted in a dose-dependent increase in neutral cholesteryl ester hydrolase activity of up to 50% (ED50 = 0.1 mM). Incubation with prostaglandin E1 in the presence of 1-methyl-3-isobutylxanthine also increased neutral cholesterol ester hydrolase activity by about 50%. In cell-free preparation, cAMP-dependent protein kinase caused about a 2-fold activation of the neutral cholesteryl ester hydrolase. Activation was blocked by protein kinase inhibitor. These data suggest that the P388D1 macrophage may be a useful model for studying the hormonal regulation of cholesteryl ester mobilization in macrophage-derived foam cells.

[23] Hormone-sensitive triglyceride lipase from rat adipose tissue

Publisher Summary This chapter discusses the determination of hormone-sensitive triglyceride lipase from rat adipose tissue. Adipose tissue contains at least two distinct triglyceride lipases. Lipoprotein lipase (LPL) is responsible for the degradation of the triglyceride moiety of circulating lipoproteins and thus controls the uptake of triglyceride fatty acids from plasma into adipose tissue. Hormone-sensitive lipase (HSL) is responsible for the degradation of stored triglycerides in response to fat-mobilizing hormones such as catecholamines, glucagon, and adrenocorticotropic hormone (ACTH). In the course of assay, free [ 14 C]oleic acid produced by hydrolysis of [ 14 C]triolein (labeled equally in all three acyl groups) is extracted and adsorbed onto an anion exchange resin. The adsorbed fatty acid is then displaced by strong base and the radioactivity is determined in a liquid scintillation counter (Method 1). Alternatively, the incubation mixture at the end of the assay period is subjected to a liquid-liquid partition to separate [ 14 C]oleic acid from unhydrolyzed [ 14 C]triolein and an aliquot of the upper aqueous phase containing the free fatty acid (FFA) is taken for scintillation counting (Method 2).

Neutral triglyceride lipase in macrophages.

High levels of neutral triglyceride lipase activity have been demonstrated In several types of macrophages (J774 cells, human monocyte/macrophages, rabbit alveolar macrophages, and resident mouse peritoneal macrophages). The pH optima ranged from 6.5 to 7.4 depending upon the buffer and the conditions of incubation. The addition of bovine serum albumin stimulated activity at low concentrations, as expected for a fatty acid-releasing reaction, but strongly inhibited at higher concentrations; maximal activity was observed In the presence of 0.625 mg/ml of bovine serum albumin. The enzyme was remarkably thermostable, showing no apparent loss of activity at 50°C for as long as 6 hours. The lipase was Inhibited 80% by 0.1 M NaCI. Assayed under optimal conditions, the specific activity of the neutral triglyceride lipase from J774 cells was more than 100-fold greater than the activity of llpoproteln lipase or neutral cholesterol esterase from those cells; this activity was 10-fold greater than the levels of hormone-sensitive lipase from 3T3-L1 adlpocytes. This neutral triglyceride lipase may play an Important role In the degradation and mobilization of cytosollc triglyceride in macrophage-derived foam cells.